Overview of the chemistry and biological activities of natural atisine-type diterpenoid alkaloids

Atisine-type C20-diterpenoid alkaloids (DAs) are a very important class of diterpenoid alkaloids, which play an important role in the biosynthesis of DAs. To date, 87 atisine-type DAs and 11 bis-DAs containing an atisine unit have been reported from five genera in two families. The genus Spiraea in Rosaceae family could be regarded as the richest resource for atisine-type DAs, followed by the genera Delphinium and Aconitum in the Ranunculaceae family. Among the reported atisine-type DAs, several possess unprecedented skeletons. Natural atisine-type DAs have a wide range of biological activities, including antitumor, antiplatelet aggregation, biological control, and anti-inflammatory, analgesic, antiarrhythmic, and cholinesterase inhibitory effects, which are closely related to their structures. In particular, the antiparasitic effect of atisine-type DAs is more prominent than that of other types of DAs, which highlights their potential in antiparasite drug discovery. In summary, the high chemical and biological diversity of atisine-type DAs indicates their great potential as a vast resource for drug discovery.


Introduction
Diterpenoid alkaloids (DAs) are a class of polycyclic nitrogencontaining natural products with complex structures that are formed by the amination of tetracyclic diterpenes or pentacyclic diterpenoids.There are more than 1500 natural DAs, primarily from plants of the genera Aconitum and Delphinium in the Ranunculaceae family and the genus Spiraea in the Rosaceae family. 1,24][5] DAs have various pharmacological effects, especially prominent anti-inammatory, analgesic, and anti-arrhythmic effects. 6rugs developed from DAs include the analgesics lappaconitine, 3-acetylaconitine, and crassicauline A and the antiarrhythmic drug guanfu-base A. 7,8 DAs possess complex polycyclic cage-like frameworks decorated with a variety of functional groups, which have attracted the sustained attention of chemists because of their complex and interesting chemical structures. 9DAs are primarily divided into four types according to the number of carbons constituting the molecular skeleton and biosynthetic pathways: C 18 -, C 19 -, C 20 -, and bis-DAs.These four categories are further divided into several or dozens of subcategories, and DAs with unprecedented skeletons are still being discovered.C 20 -DAs are second only to C 19 -DAs in terms of the number of compounds, but their chemical structures are more varied, with 9 major categories and 38 types of skeletons reported. 10mong C 20 -DAs, atisine-type DAs are a very important class of alkaloids and generally considered biosynthetic precursors of other types of DAs. 11Since the discovery of the rst compound, atisine, from A. heterophyllum (atis in Spanish) in 1896, 12 approximately 87 atisine-type DAs have been reported.Eleven bis-DAs formed by two atisine molecules or one atisine with one other DA have also been reported in recent decades.Natural atisines exhibit signicant physiological effects, including antitumor, antiplatelet aggregation, biological control, anti-inammatory, analgesic and antiarrhythmic effect, and cholinesterase inhibition effects.The high chemical and biological diversity of atisinetype DAs revealed their great potential in drug discovery.
A series of review papers and monographs on the chemical and pharmacological activities of DAs have been published, some of which include atisine-type DAs. 3,10,13However, these studies primarily focused on research involving all types of DAs, and only a small portion of the related research has been devoted to atisine-type DAs.A review on hetisine-type DAs, a group of DAs closely related to atisine-type DAs, has also been published by our group, which has highlighted their prominent antiarrhythmic effects.Previous studies have shown that atisine-type DAs exhibit different biological activities than hetisine-type DAs or other types of DAs, for example, they have outstanding antiparasitic effects.However, no individual or systematic reviews of atisine-type DAs are currently available.There have been a series of studies on atisine-type DAs in the past few decades, and signicant progress has been made.Thus, the present review summarizes the research progress on

RSC Advances
REVIEW the structural features and biological activities of natural atisine-type DAs to provide a complete overview of the existing knowledge of atisine-type DAs and promote further research and exploitation of atisine-type DAs and the utilization of related medicinal plants.

Biosynthesis
Atisine-type DAs are formed by the amination of ent-atisane-type tetracyclic diterpenoids (Fig. 1).The use of L-serine as a main nitrogen source has been supported by Hao et al. 14 Ent-atisane diterpenoids are generally biosynthesized from isopentenyl pyrophosphate (IPP), and IPP may be coproduced by the MEP (methylerythritol phosphate) and MVA (mevalonic acid) pathways. 15,16Briey, the formation of geranylgeranyl pyrophosphate (GGPP) from three IPP units is catalyzed by geranylgeranyl pyrophosphate synthase (GGPPS), and GGPP is further catalyzed to form ent-copalyl diphosphate (ent-CPP).Ent-CPP is the only precursor in the biosynthesis of all types of DAs, 17,18 and it is further cyclized to form ent-kaurane and ent-atisane, which are aminated to obtain the corresponding DAs.The pathways for the formation of DAs from the amination of other diterpenoids lack the support of intermediates found in nature, but there are more reports of ent-atisane-type diterpenoids. 19The coexistence of entatisane-type diterpenoids and atisine-type DAs in plants from Aconitum, Delphinium, and Spiraea, such as atisenol isolated from A. heterophyllum 15,20 and spiraminol from Spiraea, 14 1).The fusion of rings is identical for all atisine-type DAs, i.e., the A/ B and E/F rings are cis-fused, and the A/E and B/C rings are transfused.In addition, according to single-crystal X-ray diffraction analysis of the corresponding atisine-type DAs, such as atisinium chloride, dihydroatisine, isoatisine, atisine 15-acetoxyazomethine, and 5-hydroxyazomethine, 23,24 the six-membered cyclohexane rings A and B are in chair conformations, rings C and D are in boat conformations, and the E ring is usually in a chair conformation, but it can be distorted to a semichair conformation when an imine is formed. 25tisine-type DAs also share some common structural characteristics.For example, most atisines possess a C-16 and C-17 exocyclic double bond, while the exocyclic double bond in a few atisines might be oxidized to form exocyclic methylene groups or be reduced to the angle methyl group.Compared with other types of DAs, this type of compound possesses fewer types and quantities of oxygen-containing substituents, mainly OH, OAc, and ]O, with only 1 case of OBz (O-benzoyl) substitution and only 2 cases of 2-methylbutyryl (MeBu) substitution.Hydroxyl (OH) and OAc groups are the most common substituents on atisines, and they are usually located at C-15 or/and C-7.In addition, atisine-type DAs feature abundant substituents at the N atom, which distinguishes them from other types of DAs whose N atom is commonly substituted by an ethyl group.According to the form of the N atom, natural atisines can be divided into the amine subtype (A-1), the N,O-mixed acetal/ketal subtype (A-2), the amide/lactam subtype (A-3), the imine subtype (A-4), the quaternary ammonium salt (A-5), and the enamine subtype (A-6).In recent years, aconicatisulfonine-type rearranged atisines (A-7) and cyano-containing atisines (A-8) have also been discovered.In addition, some types of bis-DAs containing atisine units were found.Table S1 † lists the names, subtypes, plant sources, and references of a total of 87 naturally occurring atisine-type DAs and 11 bis-DAs containing atisine units reported in recent decades.Herein, the structural features of atisine-type DAs are discussed by category.
Fig. 4 The transformation of atisine and isoatisine.
Fig. 7 The quaternary ammonium salts and enamines of atisines.phenethyl substituent of this type of compound may be formed by the amination of phenylalanine when atisane-type diterpenoids are transformed to atisine-type DAs, and the cyano group may be formed by glycine via the Mannich reaction.

Bis-DAs containing an atisine unit
Bis-DAs are DA dimers formed by the condensation of two C 20 -DAs or one C 19 -DA and one C 20 -DA (Fig. 10).Twenty-three bis-DAs have been found in nature, 11 of which contain an atisine unit in their structures.These compounds can be further divided into 4 subtypes according to the construction units, namely, the atisine-denudatine subtype, the denudatine-atisine subtype, the hetidine-atisine subtype and the hetidinerearranged atisine subtype.Atisine-denudatine-type bis-DA is formed by linking atisinetype DA and denudatine-type DA via a C-17-O-C-22 0 ether bond.
Bulleyanine B ( 88) is an atisine-denudatine bis-DA isolated from A. bulleyanum and is the only member of its subtype reported thus far. 54The denudatine-atisine type is created by the formation of an ether bond between C-17 in the denudatinetype DA and C-22 0 in the atisine-type DA.Two compounds of this type have been reported, piepunine (89) isolated from A. piperunense 55 and bulleyanine A (90) isolated from A. bulleyanum. 54Hetidine-atisine-subtype bis-DA is formed by linking C-16 and C-17 in a hetidine-type DA with C-15 0 , C-16 0 and C-17 0 in an atisine-type DA to form a tetrahydropyran ring.Only two compounds of this subtype of bis-DAs have been reported, staphisagrine (91) and staphisagnine (92), from D. staphisagria. 56The connection pattern of hetidine-rearranged atisinetype bis-DAs is analogous to that of hetidine-atisine-type bis-DAs.The difference is that the A ring in the atisine unit is rearranged, the C-20-C-10 bond is rearranged to C-20-C-2.There are six components (93-98) in this category, all of which were isolated from D. staphisagria. 57,58

Distribution
Atisine-type DAs are distributed in plants of two families and ve genera, namely, Aconitum, Delphinium, Consolida, and Thalictrum in the Ranunculaceae family and Spiraea in the Rosaceae family (Table 1).Only eight atisines have been found in Consolida, 28,[59][60][61] and only one atisine-type DA has been isolated from Thalictrum. 62Spiraea are the richest source of atisines, with a considerable number coming from S. japonica.Thirty-three atisines have been isolated from the genus Delphinium.Among the 11 bis-DAs that have been isolated, 8 were isolated from D. staphisagria.
In terms of the distribution of individual compounds, most atisine-type DAs were reported from only one certain species.This means that these compounds may be used as chemical markers for this plant to assist in its taxonomic identication.The most widely distributed compound is atisine (21), which has been isolated from 26 species.Ajaconine (15) was found in 16 species.However, they were all isolated from Aconitum and Delphinium.These two compounds may be precursors of other atisine-type DAs and other types of DAs, such as C 19lycoctonine-type DAs, in Aconitum and Delphinium plants. 63

Antitumor activity
Some atisine-type DAs signicantly inhibited tumor cell proliferation in vitro.Honatisine ( 27) isolated from D. honanense inhibited the proliferation of the human breast cancer cell line MCF-7, with an IC 50 value of 3.16 mM, which was better than that of the positive control etoposide (IC 50 , 7.53 mM). 37Delphatisine C (25) isolated from D. chrysotrichum showed signicant in vitro tumor cytotoxicity against the human lung adenocarcinoma cell line A549, with an IC 50 of 2.36 mM, which was comparable to that of the positive control etoposide. 36Li et al. reported that the cyano-containing compounds brunonianines A-C (85-87) inhibited the proliferation of Caco-2, H460 and Skov-3 tumor cells (Table 2).Brunonianines B and C (86 and 87) inhibited the proliferation of Caco-2 cells to a level comparable to that of the positive control hydroxycamptothecin, while 86 had a stronger inhibitory effect on the proliferation of Skov-3 cells than the positive control.The difference in the antitumor activities of brunonianines A and B can be attributed to the stereochemistry of the cyano-containing C-19, with the S conguration being signicantly more potent than the R conguration.Further studies revealed that brunonianine B (86) arrested the Skov-3 cell cycle in the G2/M phase, reduced the mitochondrial membrane potential, and induced the apoptosis of Skov-3 cells to inhibit cell proliferation.This antitumor effect may be partially due to its inhibition of cell movement to prevent tumor metastasis.Western blot analysis revealed that it induced cell apoptosis via the Bax/Bcl-2/caspase-3 signalling pathway. 53ao et al. performed in vitro antitumor activity studies on 11 structurally modied derivatives (S1-S11) of spiramines C and Review RSC Advances D (34 and 35) (Fig. 11). 64These compounds inhibited the proliferation of a series of tumor cells, including HL-60, SMMC-7721, A-549, MCF-7, and SW-480 cells (Table 3).The toxicity of the S1 and S2 derivatives against the ve tumor cell lines was greater than that of the S-3 and cisplatin positive controls, which showed the potential for their development as anticancer drugs.All the derivatives induced the apoptosis of Bax/Bak double knockout murine embryonic broblasts (Bax −/− /Bak  4) had signicant inhibitory effects on the proliferation of wild-type MCF-7 cells and adriamycin (ADR)-induced multidrug-resistant MCF-7 cells (MCF-7/ ADR), which indicated that spiramine derivatives may overcome drug resistance.Preliminary structure-activity relationship studies showed that the oxazolidine ring was essential for cytotoxicity (Fig. 12).However, the C-7-O-C-20 ether bond is unfavorable for cytotoxicity.Atisine derivatives bearing double "Michael response receptor" groups outperformed derivatives with mono "Michael response receptor" group in inducing the apoptosis of Bax −/− /Bak −/− cells and cytotoxicity in tumor cell lines.

Anti-platelet aggregation effect
Thrombotic disease is a common cardiovascular disease that seriously harms human health. 65Platelet aggregation is a key factor in the development of thrombi, and the discovery of antiplatelet aggregation components from natural products is highly important. 66Hao et al. reported that the atisine-type DA spiramine Q (43) had selective anti-arachidonic acid-induced platelet aggregation activity, and its inhibitory effect was stronger than that of aspirin. 67Hao et al. subsequently evaluated the antiplatelet aggregation effects of six atisine-type DAs and their eight structurally modied derivatives on the basis of arachidonic acid, adenosine diphosphate (ADP) and plateletactivating factor (PAF). 67,68 Among the atisine-type DAs tested,  12 compounds (Table 5) signicantly inhibited PAF-induced platelet aggregation in a concentration-dependent manner but had no effect on the aggregation induced by ADP or arachidonic acid, which showed selective inhibition.The antiplatelet aggregation effects of hetisine-type DAs, which are structurally similar to atisine-type DAs, are poorly selective. 69Among the screened compounds, only spiramine C1 (Fig. 12) concentration-dependently inhibited platelet aggregation induced by PAF, ADP, and arachidonic acid, which indicated a nonselective antiplatelet aggregation effect.The inhibitory effect of spiramine C1 on arachidonic acid was comparable to that of aspirin.Preliminary SAR analysis (Fig. 11) showed that an oxazolidine ring was critical for their antiplatelet aggregation effect.The antiplatelet aggregation effect of atisines was strongly affected by substitution at C-15.Atisine-type DAs are a novel class of selective antiplatelet aggregation agents.Although they are less potent than the known PAF receptor-specic antagonist ginkgolide B, more effective derivatives may be obtained via structural modication.

Biological control effects
Some plants of the genera Aconitum and Delphinium may be used as pesticides, which supports the potential biocontrol properties of these main ingredients.Plant virus diseases are a group of important agricultural diseases whose harm to cultivated crops is second only to fungal diseases.Tobacco mosaic virus (TMV) is a model virus for plant virus research, with a wide range of host plants and a worldwide distribution.It can infect a variety of important economic crops, such as tomato, potato, pepper, cucumber, and tobacco, and cause major economic loss. 70The discovery of active substances against TMV has important practical signicance.Hao et al. used the half-leaf method to screen a series of atisine-type DAs isolated from S. japonica var.acuminata for anti-TMV effects, including protective effects and healing effects.The experimental results showed that most of the compounds exhibited Fig. 12 The structures of derivatives of spiramines C and F (C1-C4, F1-F4).Review RSC Advances greater activity than did the positive control ningnanmycin.The inhibition rates of some compounds, such as 38, 65, 67 and 68 (Table 6), were 73.5-92.9% at 100 mg mL −1 .Preliminary SAR analysis showed that atisine-type DAs with an imine at C-20 and an acyl substituent at C-6 had better antiviral effects.Compounds 4 and 47 also exhibited healing effects that were superior to those of ningnanmycin.Reina et al. studied the insecticidal effects of more than 70 DAs, including nine natural atisines and two synthetic derivatives called alkaloids A and B. 71,72 As shown in Table 7, the antifeeding effect of the satisfactory-type compounds was structure-and species dependent.Except for compounds 48, 50 and 61, most of the atisines tested had antifeedant effects on the potato beetle Leptinotarsa decemlineata (CPB), with EC 50 values ranging from 3.9 to 6.9 mg cm −2 .Only six atisines showed antifeeding effects on S. littoralis, with EC 50 values ranging from 0.1 to 8.2 mg cm −2 .In general, the antifeeding effect of atisines was weaker than that of C 19 -DAs.Compound 48 exhibited the strongest antifeeding effect on S. littoralis (EC 50 of 0.1 mg cm −2 ), but it was ineffective against CPB.Compound 3, which had a strong effect on CPB (EC 50 of 2.9 mg cm −2 ), was also effective against S. littoralis.Reina et al. further tested the effects of DAs on insect Sf9 and CHO cells and Trypanosoma cruzi epimastigotes, which are two biological models lacking neurotransmission.Compounds 48 and 21 exhibited Sf9 cytotoxicity, and compound 50 exhibited toxicity against T. cruzi epimastigotes.These toxic effects suggest that the mechanism of action of atisines is different from that of C 19 -DAs.C 19 -DAs have strong neurotoxicity and specically act on voltage-gated Na channels.SAR analysis revealed that the antifeedant effect of atisines on CPB was related to oxygen substitutions at C-9 and C-15, and the presence of a ketone group at C-15 resulted in a selective CPB antifeedant (e.g., compounds 16 and 17).Atisines with OH-15 or OAc-15 substituents and without OH-7 substituents had antifeedant activity against S. littoralis.Imines also enable atisines to exhibit antifeedant effects on S. littoralis (Fig. 13).
These DAs have also been screened for their antiparasitic effects (Table 8), and only three atisine-type Das, 50, 61 and 76, exhibited strong in vitro leishmanicidal activity against the promastigote L. infantum.Among these DAs, 76 had the strongest effect, followed by 61, and 50 had the poorest effect. 73  Fig. 13 The SAR of atisine-type DAs.
epimastigotes.Atisinium chloride showed a strong inhibitory effect similar to that of the reference drug benznidazole.These compounds also attenuated the ability of the parasite to invade mammalian cells, and its ability to replicate and transform into trypomastigotes in the cells and were nontoxic to host cells (Vero cells) (compound 21, IC 50 > 300).These studies highlight the ideal insecticidal effect of atisines, and their signicant effect and unique molecular selectivity are worthy of further investigation.The use of atisine-type DAs as lead compounds via structural modication will likely result in compounds with stronger activity and provide new medicinal resources for the prevention and treatment of protozoal infections.

Anti-inammatory effect
Plants of the genera Aconitum, Delphinium, and Spiraea are widely used to treat inammation-related diseases, such as arthritis and pain, which suggests that their main components, DAs, have anti-inammatory effects.

Cholinesterase inhibition
Ajaconine (15) isolated from D. chitralense inhibited cholinesterase with IC 50 values for AChE (acetylcholinesterase) and BchE (butyrylcholinesterase) of 12.61 mM and 10.18 mM, respectively. 80A mechanism-based kinetic study showed that it was a competitive inhibitor of AChE and BchE.Heterophyllinine-B (31) isolated from A. heterophyllum from Turkey had selective cholinesterase inhibition with an IC 50 value of 40.63 mM for BchE inhibition but no inhibitory effect on AChE. 81

Conclusion
A total of 87 atisine-type DAs and 11 bis-DAs containing atisine units have been reported from ve genera in two families.The genus Spiraea in the Rosaceae family may be the richest resource for atisine-type DAs, followed by the genera Delphinium and Aconitum in the Ranunculaceae family.Among the reported atisine-type DAs, several possess unprecedented skeletons.Atisine-type DAs have a wide range of biological activities, including antitumor, antiplatelet aggregation, biological control, anti-inammatory, analgesic, antiarrhythmic, and cholinesterase inhibitory effects.The antiparasitic effect of atisine-type DAs is more prominent than the other types of DAs, which highlights their potential in antiparasitic drug discovery.In summary, the high chemical and biological diversity of atisine-type DAs indicate their great potential as a vast resource for drug discovery.

Fig. 1
Fig.1The structures of atisine-type DA and its closely related DAs.

3. 2 .
The N,O-mixed acetal subtypeThe N,O-mixed acetal compounds are the most numerous atisine-type DAs, with 33 compounds identied.These compounds may be further divided into three classes according to the type of N,O-mixed acetal group (Fig.3).The rst class of compounds includes six compounds(15-20) that feature an N-C-20-O-C-7 group.32,33Among these compounds, compound 19 possesses a rare OMeBu substituent and compound 20 is the only atisine-type DA possessing an OBz substituent.34The second class of 11 compounds(21-33) feature an oxazolidine ring, which may be formed by N-C-21-C-22-O-C-20 or N-C-21-C-22-O-C-19.

©
2024 The Author(s).Published by the Royal Society of Chemistry RSC Adv., 2024, 14, 22882-22893 | 22885 Review RSC Advances carmichaelii. 51The structure of compound 82 was veried using single-crystal X-ray diffraction.These two compounds are amphoteric compounds with sulte and quaternary ammonium groups.Compounds 82 and 83 have a unique rearranged sevenmembered C-ring, which may be the result of SO 3 H attacking the C-16 of atisine with a C-16-O-C-17 epoxy group, followed by Wagner-Meerwein rearrangement. 10 Barpuberudine (84) has a rearranged novel atisine skeleton and was found in A. barbatum var.puberulum. 26Barpuberudine (84) features a C ring in which the C-11-C-12 bond is broken instead of a C-11-O-C-15 linkage, which forms a tetrahydrofuran ring.Barpuberudine (84) possesses a spiro D ring with a rearranged D 12,16 group.Notably, anthriscifolsine A is a hetidine-type DA with a similar rearranged C/D ring, and it was isolated from D. anthriscifolium var.majus. 523.7.The cyano-containing subtype Brunonianines A-C (85-87) (Fig. 9) containing an unprecedented cyano substituent at C-19 were isolated from D. brunonianum. 53Brunonianines A and B (85 and 86) are a pair of epimers, and the structure of brunonianine A (85) was conrmed using singlecrystal X-ray diffraction.Brunonianines A and B (85 and 86) have a phenethyl substituent on the N atom, and brunonianine C (87) has a p-hydroxyphenethyl group on the N atom.The

Fig.
Fig. The rearranged subtype of atisines.

−
MEFs) to different degrees, while S1, S2, S9 and S11 exhibited the strongest activities.Their IC 50 values were 2.243 mM, 3.377 mM, 4.524 mM and 1.814 mM, respectively, and the IC 50 of the positive control S-3 was 1.736 mM.The induction of Bax-and Bak-decient cells and cancer cell apoptosis by S1, S2, S9 and S11 was also veried by annexin V/PI and caspase activation experiments.Compared to the positive control adriamycin, compounds S2, S3 and S6 (Table
Reina et al. used an in vitro culture system of mammalian host cells (Vero) infected with Trypanosoma cruzi to screen anti-T.cruzi agents from a total of 43 C 19 -DAs and 21 C 20 -DAs. 74Among the 64 DAs tested, only ve C 20 -DAs were active against T. cruzi

Table 3
The IC 50 values of cytotoxicity of spiramine derivatives (mM)

Table 5
The anti-platelet aggregation effect of atisine-type DAs (IC 50 , mM)

Table 7
Antifeedant activities of atisine-type DAs